The primary goals of this laboratory are to understand the basic mechanisms by which trace elements are absorbed, transported, assimilated, stored and excreted, and then to apply this knowledge more effectively for the detection and prevention of pathological conditions induced by nutritional deficiencies. Two distinct but related research strategies for our future trace element research are: 1) The regulation and control of intracellular redox and assimilation of Fe(III) and Cu(II) by heme proteins in a variety of cells and tissues with particular concern for interactions among the trace elements; 2) The mechanisms by which deficiencies of Mn, Cu and Zn affect the physiological, cellular and biochemical processes of bone metabolism. Hemoglobin and myoglobin are potent reducing agents for Fe(III) and Cu(II). The exploration of mechanisms by which these proteins carry out intracellular reduction, the characterization of heme proteins that may be operative in other tissues, such as cytochrome P450 or b5, and the definition of the metabolic role for such systems in vivo will be investigated. High resolution NMR and stopped-flow spectrophotometry will elucidate the protein binding sites and the kinetics of reduction of Fe(III) and Cu(II) chelates. Amino acid sequences of diverse species of myoglobin and of human hemoglobin mutants will provide altered binding or reduction of the metals in vitro, and will be correlated with altered ferrokinetics in vivo. The mechanism by which Cu(II) enhances the rate of Fe(III) reduction by hemoglobin will be defined and compared with the cellular events induced by Cu-deficiency anemia. The ability of other metals to inhibit or enhance Fe and Cu reduction will be related to the known influence of these metals on iron metabolism. A model of osteopenia has been developed in rats fed a Mn-deficient diet for 12 months. Supplementation of these deficient animals with Mn following the appearance of bone pathology will be tested in a similar fashion. Ectopic subcutaneous implantation of demineralized bone powder to measure osteoblast activity and bone powder (organic and mineral matrix) to estimate osteoclast activity will be carried out in normal and deficient animals. The kinetics of 45Ca in normal and Mn-deficient animals will be analyzed. Changes in the organic and mineral matrix of the bones will provide information concerning enzymic deficiencies and/or alterations in the structure of the mineral. Techniques developed in this lab for the quantitation of trace element and calcium status in experimental animals will be applied to a series of normal and osteporotic human sera.